Apparatus for causing medicinal products to penetrate into red blood cells

ABSTRACT

An apparatus for incorporating one or more biologically active substances into red blood corpuscles (erythrocytes) by lysis and resealing technique comprises a washing unit, a lysis unit and a resealing unit. The washing unit obtains a suspension of erythrocytes from complete or incomplete blood. The lysis unit cools the erythrocyte suspension to a temperature below 10° C. and maintains this temperature while the suspension is processed through a dialysis unit and subsequently exposed to the active substance. The treated erythrocyte suspension is held in a retardation pouch for a predetermined time and thereafter fed to the resealing unit, which heats the treated suspension to a temperature higher than 20° C. and maintains this temperature while the treated suspension is exposed to the resealing product. The suspension exposed to the resealing product is collected in a collecting pouch for a predetermine period before it is discharged to the washing unit.

BACKGROUND

The present invention relates to a device permitting the implementationof the so-called "lysis-resealing" technique which makes it possible toincorporate active ingredients into red blood cells.

The lysis-resealing technique is described in patents EP 101341, U.S.Pat. Nos. 4,752,586 and 4,652,449, and its principle will not beredescribed in detail.

In the so-called "lysis-resealing" technique, the primary compartment ofa first dialysis element is continuously supplied with an aqueoussuspension of erythrocytes, the secondary compartment containing anaqueous solution which is hypotonic relative to the suspension oferythrocytes in order to lyse the erythrocytes. The erythrocyte lysateis then in contact with the substance exhibiting a biological activityand in order to reseal the erythrocyte membrane, the osmotic and/oroncotic pressure of the erythrocytic lysate is increased after bringingit into contact with the biologically active substance.

In this preferred embodiment of this process, the resealing is performedin a separate vessel although it is possible, as described in thepatent, to carry out the resealing using successively two dialysisbobbins.

More specifically, in this type of process, the globular pellet isobtained by centrifugation and decantation of plasma, which is kept at4° C. for the treatment. The red blood cells are subjected to a firstseries of washes. The first wash in physiological saline makes itpossible to obtain a globular concentrate whose buffy coat (white bloodcells, platelets) is removed. The concentrate of red blood cells is thenbrought into contact with IHP (inositol hexaphosphate) for exampleduring the subsequent two washes.

In hypotonic medium, the red blood cell swells up to a volume which maybe equal to 175% of its initial value (ROPARS et al., 1986). It is atthis stage that pores of a few hundred angstroms appear. There is thenexchange between the extracellular and intracellular media especiallyfor the substance to be internalized. After addition of a hypertonicsolution to the hemolysate, the isotonicity is restored, the poresbecome closed again and entrap the IHP for example inside the red bloodcell. An incubation phase, which is necessary for the charged red bloodcells to recover permeability characteristics identical to those of theinitial red blood cells, follows in regenerating solution. Afterresealing, the red blood cells are subjected to a second series ofwashes. The first two washes use a solution of physiological saline:they make it possible to remove the non-resealed red blood cells.

The red blood cells are then resuspended in autologous plasma to aphysiological hematocrit which permits transfusion. The converted unitof blood consists of red blood cells with improved oxygen binding andtransporting properties whose morphological and physiologicalcharacteristics are similar to unconverted red blood cells.

The implementation of the lysis-resealing process permits theincorporation of a large number of active ingredients into red bloodcells, which red blood cells can be reinjected into the same patient orinto different patients. For example, the incorporation of IHP into theerythrocytes makes it possible to modify the affinity constant ofhemoglobin for oxygen. Other applications of this process are describedin the patents mentioned above.

SUMMARY

The present invention relates to a device intended to allow theincorporation of one or more biologically active substances into redblood cells by the lysis-resealing technique. It comprises

a washing unit which permits, starting with a blood, whole or otherwise,a suspension of red blood corpuscles to be obtained; and

a lysis and resealing unit consisting of a lysis module at a temperatureless than 10° C. and a resealing module at a temperature greater than20° C., all the elements of the lysis and resealing unit entering intocontact with the suspension of erythrocytes being designed for a singleuse.

"Designed for a single use" should be understood to mean that even whenthe elements are intended to be reused, they will be reused only aftersterilization and/or viral inactivation but will be designed as elementsfor single use, that is to say capable of being easily assembled anddismantled and of low cost.

In the device of the present invention, the lysis module A comprises

a set ensuring the transport of a lysis buffer (2) up to the inlet ofthe dialysis cartridge and the optional discharge (3) of the said lysisbuffer, as well as the elements which make it possible to maintain themodule at a determined temperature, especially 4° C.,

a removable set preferably for single use, incorporating a dialysiscartridge (9) capable of being attached to the lysis buffer feed inorder to supply one of the compartments, the other compartment of thecartridge being connected at the venous end to a vessel (6) intended foradjusting the temperature of the erythrocyte suspension, and at thearterial end to a storage vessel (11).

The resealing module comprises a set of elements which allow the moduleto be maintained at a determined temperature, preferably 37° C., and aremovable set, preferably for single use and incorporating from upstreamto downstream, a vessel (22) intended to ensure adjustment of thetemperature of the suspension, connected to a vessel (26) incorporatingan access for resealing solution (28).

These modules as a whole incorporate a pump which permits the entry ofthe erythrocyte suspension into the lysis module, a pump which permitsthe exit of the erythrocyte suspension from the resealing module, and anintermediate pump ensuring the circulation between the lysis module andthe resealing module. These pumps are preferably peristaltic pumps,which avoid all contact with the suspension.

The device preferably incorporates, in addition, before the resealingunit, a small tube for admitting a solution of an active ingredient tobe internalized, for example ATP (adenosine triphosphate). For theinternalization, depending on the substance to be internalized, thelatter may be introduced with the erythrocyte suspension before thelysis (case of IHP), or alternatively added after the lysis at 4° C.(case of ATP). The vessels intended to ensure the adjustment of thetemperature of the suspensions are preferably plastic pouchesincorporating internal baffles. These preferably elongated pouches areplaced vertically and supplied via the top and discharged via thebottom.

The heating and cooling of the modules can be ensured by a support platewhich will receive, on one face, the elements for single use and theother face will be in contact with a cooling or heating element.Likewise, in the case of the lysis module, the lysis buffer will becooled by passage into an appropriate cooling system.

The dialyzer itself can be used as heat exchanger between the blood andthe lysis buffer which pass through it, without resorting to otherartifices. These heat exchanges permit a specific treatment of the redblood corpuscles.

The various modules placed on these plates, whose form will bepreferably adapted in order to attach the removable elements, will beable to receive a cover and a coating which will ensure that thetemperatures are preferably maintained at 4° C. and 37° C.

The washing element may take various forms; it will be preferably of theseparating bowl type, it will also be possible to reuse it to perform awashing after treatment of the suspension.

The washing element is, in its principle, a reservoir having arotational symmetry and comprises two casings in the form of concentrictruncated cones, the separating chamber itself being the space betweenthe two conical surfaces.

One of the characteristic features of the separating bowl is that itsfilling is performed at its base and at its periphery; any productintroduced, if it is less dense than those already present in theseparating chamber will therefore have to circulate in the directionopposite to sedimentation.

The components separated from the product introduced become organized inconcentric rings and become concentrated through the gradual decrease intheir diameter and the decrease in their height by the conicity of theseparating chamber.

The various fluids circulate and make their way towards this bowl byvirtue of a peristaltic pump and eight pneumatic clamps. An airdetector, two optical sensors and two coder wheels placed, one over theaxis of the pump, and the other over the axis of the centrifuge, permitthe collection of information necessary for the management of theperipheral units as a whole which are the pump, the centrifuge and theclamps.

Elements with the same type of functions are already commercializedespecially by the company Haemonetics on the machines V50/PCS/CELLSAVER®, for example the washer COBE 2997, the separators COBE Spectra,DIDECO Vivacell, BAXTER CS 3000 and Autopheresis PC.

The description below represents an embodiment of the device accordingto the invention, using preferably a separator from the companyHaemonetics V50®.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general schematic representation of the device for singleuse.

FIG. 2 is a schematic representation of the separating bowl.

DETAILED DESCRIPTION

The lysis-resealing device represented in FIG. 1 incorporates a lysisunit A and a resealing unit B.

The lysis unit A comprises of an evaporator which is in contact with astainless steel metal plate (1) having the form of the elements of thekit for single use. The other side of the evaporator (not represented)is in contact with the non-disposable heat exchanger containing metalcoil. In operation, a second metal plate which is cooled oralternatively simply made from insulating material joined to the firstencloses the kit for single use in a minimum cooled volume. This boxthus constituted dictates, in addition, the maximum volume of thevarious pouches and small tubes.

This metal plate (1) incorporates two elements permitting the arrival(2) and departure (3) of the lysis buffer, of which the supply system isnot represented, but consists essentially of a pump, a cooling circuitand a supply tank. On the plate (1) is removably attached the part ofthis lysis module called kit for single use. This part is composed, fromthe blood inlet to the outlet of the blood treated: of a perforator (4)followed by a small PVC tube (5) which opens out into a special pouchwelded in the form of a coil (6) and acting as heat exchanger; a 1 to1.5 m coil made from a Stainless Steel tube 1/8 OD could be used inplace of the coil-shaped PVC pouch for the heat exchanges with theblood. The other end of this exchanger is itself linked to a small tube(7) which is connected via a special male luer to the arterial inlet (8)of the dialysis cartridge (9). At the venous outlet (10) of thedialyzer, a small tube (12) leads to the top of a 600 ml PVC pouch (11)which serves as buffer volume, this small tube is joined by another toform a "Y" (13), between the outlet of the dialyzer (10) and the"retardation" pouch (11). Finally, a last small tube (14) leads from thebottom of this pouch to the outlet of the module. The lysis buffer inletof the dialyzer comprises a small tube passing across a peristaltic pumpand leading to an exchanger formed by a non-disposable metal coilmounted on the evaporator. The outlet of the dialyzer, as regards thedialysis liquid, is for its part a simple small tube opening out into areservoir. The elements to be cooled are therefore overall of two types,on the one hand the kit for single use made from plastic material (PVC,polycarbonate) and the reusable metal kit both in intimate contact withthe thermoregulated metal plate, and on the other hand the fluids(blood, lysis buffer, solution of active ingredient) which circulate andare held in the kit in whose contact acquire the desired temperature.

The lysis module also comprises a pump small tube (15) for additivesolution which can be mixed with the blood lysed at 4° C., for exampleATP or a small molecule. This small tube is linked to the small tube(7). Two levels are therefore possible for bringing the active substanceand the red blood cells into contact, before and immediately after thelysis.

Finally, the blood circulation is ensured by a peristaltic pump (16)which is directed by a set of controls like the other peristaltic pumpsof the device. The pump (17) fitted onto the small tube (18) is linkedto the "Y" (13) and is essentially intended to ensure the purge of thedialysis bobbin (9). This pump (17), like the previous one, is directedby the central control unit.

The delivery of active ingredient by the small tube (15) can be achievedeither manually, or via a pump (not represented).

The resealing module B is itself also made up of a metal box (20)provided with a port (not represented) which closes over a kit forsingle use. This metal box, upon contact with a heat source, will ensurethat the module in question is maintained at 37°. The kit for single useis composed, from the inlet for the blood at 4 degrees to the outlet ofthe heated blood, of a small tube (14) coming directly from the "4degrees" module, and which permits preheating of the blood. This smalltube temporarily leaves the module and passes into the body of a bloodpump (21) before joining a heat exchanger (22) identical to that used inthe 4 degrees module. The small tube (23) which leaves the exchanger,placed for its part in the 37 degrees module, joins a "Y" (24) one ofwhose branches ends with a male perforator (25) (to be connected to theresealing bottle). The other branch of the "Y" leads to 600 ml PVC pouch(26). This pouch has two opposite small tubes for access (27 and 28),permitting the first blood to enter to be also the first to leave, andis identical to the "retardation" pouch of the "4 degrees" module. Asmall tube ending with a female mouthpiece (29) for the perforatorconstitutes the outlet of this "retardation" pouch.

Finally, the admission of the resealing solution which is achieved viathe small tube (25) and the pump (30) is also controlled like theprevious peristaltic pumps at the central control unit. As can beobserved on reading this figure, the set of small tubes for the fourpouches (6), (11), (22), and (26) and the dialysis bobbin (9), which arethe elements in contact with the suspension of red blood cells, areremovably placed in the two resealing units, and can consequently eitherbe resterilized or alternatively discarded after use.

The pouches for adjusting the temperature are made from PVC andcomprises two practically rectangular and identical PVC sheets weldedtogether in the form of a coil. This coil leads from a small PVC tube(5) situated at the top of the pouch, to a second tube (7), which iswelded to the bottom of the pouch.

Inside the closed box in which the pouch is enclosed, the maximumdistance between the inner faces of the pouch will be fixed and of theorder of few tens of mm. In this manner, the exchanger volume will besmall (from 10 to 20 ml) and the surface in contact with the plate (1)at 4 degrees will be maximum.

The dialysis cartridge ( 9 ) incorporates a parallelepipedic box madefrom rigid plastic material. This box is separated into two compartmentsby semi-permeable membranes, each of these compartments has twoinlet-outlet tubes. One of the compartments is crossed by blood, theother by lysis buffer. The assembling of this element in the module isperformed such that blood circulates from the bottom to the top so as toensure a natural purge of the blood compartment.

The dialysis cartridge may also be of the "hollow fiber" type, where thetwo compartments comprises, for the blood compartment, the inside of thehollow fibers (made from semipermeable materials) and for the lysisbuffer compartment, the volume outside the fibers and inside the rigidbox enclosing these fibers.

The dialysis outlet buffer pouch (11) is a transfer pouch with a maximumvolume of 600 ml, enclosed in a volume of about 500 ml in which itincreases in volume during its filling. It has the special feature thatthe small tube for supplying and that for discharging are both mountedon a short side of the pouch, but in an opposite manner. For a verticalposition of the pouch, the first blood to enter (via the top) will alsobe the first to leave (via the bottom).

FIG. 2 represents the section of the washing unit, the description ofthis separation bowl will not be made in detail since it is a productwhich can be found on the market and which was adapted for the presentuse.

In the sectional view of FIG. 2, it can be seen that the separating bowl(41), in its principle, consists of two rigid casings in the form ofconcentric truncated cones (42) and (43). The first is external (42) andthe second (43) is placed on the same axis and included in the first.The separating chamber (44) is the space between the two conicalsurfaces. This bowl is provided with an injecting system (45) and acollecting system (46), separated from the separating chamber by arotating joint (47) at the vertex of the two truncated cones.

The reservoir, during the separation of the cells from the introducedsuspension, is set into rotation by a centrifuge, the injecting andcollecting system is for its part maintained by two articulated arms inthe closed position.

The injector (45), via its connection to a network of small tubes,permits the introduction, into the separator (44), of the cellsuspension to be separated, or, during stoppage of the bowl, theemptying thereof.

The collector (46), as for its part, permits the collection of theseparated constituents, which are presented to it sequentially, pushedoutwards by the introduced suspension. The collector too is connected toa network, which amounts to a small tube which joins a waste pouch.

The solid elements of the suspension are gradually concentrated bycentrifugation towards the outside of the chamber (44). A detectionsystem, previously calibrated before the centrifugation, makes itpossible, when the concentration of red blood cells has reached adetermined volume, and/or when the red blood cells have beenappropriately washed, to obtain a suspension of red blood cells havingthe desired characteristics.

The examples below are intended to demonstrate other advantages andcharacteristics of the present invention, but do not limit it in anymanner.

EXAMPLE I Separation of Red Blood Corpuscles from Plasma

The separation of red blood corpuscles is performed during the firstwash, called wash I, and permits the treatment of the blood, before thestep of reversible lysis of the red blood corpuscles and theirresealing.

The wash I, performed on a unit of whole blood, comprises the followingsteps:

removal of plasma,

removal of leucocytes

removal of platelets

adjustment of the hematocrit

suspension of the red blood corpuscles in a solution of product to beinternalized (IHP).

control of the osmolarity.

These operations which are currently manual, are performed automaticallyby virtue of the present invention. When a large quantity of blood needsto be treated, this wash may comprise several cycles.

Sequence of Operations

A cellular suspension of about 40% hematocrit is prepared by addition ofphysiological saline from an erythrocytic concentrate, this beingautomatically and simultaneously to the introduction of the erythrocyticconcentrate into the bowl, by alternating a quantity of water and bloodin a proportion which is calculated by the machine so as to obtain 40%hematocrit.

The diluted pellet is introduced into the bowl described in FIG. 2 untiloptical detection or vacuity of the pouch is achieved, a volume of about480 cc, at an output of the blood pump of 100 cc/min (adjustable from 0to 250 ml/min) and a centrifuge rotation speed of 6000 rpm. Thesupernatant solution is collected in a 5 liter waste pouch. It is duringthis step that the hematocrit is empirically determined by stoppage ofthe filling of the separator at a determined supernatant/cell interfacelevel (42 ml), this under precise output and centrifugation conditions.

The washing of the red blood corpuscles is performed by introductioninto the bowl of a programmed volume of 9% NaCl solution, this at avariable output, under the control of the optic of the small tube. Theoutput of the pump is controlled in such a manner that the liquidleaving the bowl is at a hematocrit of the order of 1%, allowing theremoval of the buffy coat while minimizing the loss of red blood cells.This step relates to the completion of the plasma removal, as well asthe removal of the platelets and the white blood cells.

The blood now being in the form of a suspension of "pure" red bloodcells in the washing solution, the suspension in IHP solution isachieved by repetition (four times) of the following procedure:

a small volume of IHP is introduced into the bowl (of the order of 50 mlto 38 mM/l of IHP), which displaces the washing solution towards thewaste pouch, then the centrifuge is stopped so as to homogenize the IHPin the solution of suspension of red blood cells, immediately followedby restarting of the centrifuge and a waiting period of about one minutein order to obtain correct sedimentation.

When the number of desired washes is achieved, the emptying of the bowlinto a transfer pouch (150 ml/min) can then take place.

In the case of the volume of blood to be treated is not sufficient toobtain a hematocrit of 72% before the removal of leucocytes in theseparator (265 cc bowl) that is to say if the volume of red bloodcorpuscles is less than 191 ml, the machine will alarm (detection ofair). The user will have in this situation the choice between 3alternatives:

continue the washing with an abnormal hematocrit

connect another unit and continue the filling of the bowl normally

if no other blood apart from that already in IHP is available, thelatter is used to complete the filling of the bowl to 72% hematocrit.The washing can then be completed as described above.

The wash I is then finished and the dialysis phase can start.

EXAMPLE II Lysis and Resealing Step

This lysis and resealing phase occurs after the wash I and thereforeinvolves a suspension of red blood corpuscles (70%) in an IHP solutionof variable volume, of 265 cc minimum to about 1.3 liters. Thissuspension will be subjected to the following operations:

reduction of the temperature to 4° C.

passage into the dialyzer

mixing with an ATP solution

waiting at 4 degrees for about 10 minutes

heating to 37° C.

mixing with the resealing solution

waiting for the wash II at 37° C. for about 30 minutes.

Sequence of Operations

The suspension of red blood corpuscles coming from the wash I is carriedby a peristaltic pump (16) through a heat exchanger (6) where itstemperature decreases to 4° C. This output is adjustable from 0 to 32cc/min in steps of 1 ml/min, and determines the blood transit time inthe dialyzer (9) for a given dead volume of the blood compartment. Thetemperature, for its part, is adjustable from 1° C. to room temperature.This pumping operation is carried out in an uninterrupted manner untilthe pump inlet air detector is activated, indicating the vacuity of thepouch of blood to be treated. As this very moment, and in order to allowthe emptying, even partial, of the heat exchanger, then of the dialysiscartridge, sterile air will be pumped for a fixed volume.

The red blood cells, once cooled in the heat exchanger (in suspension inthe IHP solution) cross the blood compartment of a dialyzer at theoutput dictated by the user's program. The temperature of the dialyzeris controlled (4° C.). The transmembrane pressure is measured. Theoutput of the lysis buffer pump (not represented) is regulated so as tomaintain the transmembrane pressure constant and at the desired level,from 0 to 300 mmHg. The lysis buffer is itself maintained at 4° C. bypassage through a nondisposable heat exchanger.

At the outlet of the dialyzer, and still at 4° C., the cellularsuspension obtained is proportionally mixed by means of the small tube(17) with an ATP solution, adjustable from 1/20th to 1/5th (ratiopreadjusted to 1/10th), then allowed to stand for a maximum of 15minutes, (period adjustable from 0 to 20 min) in the pouch (11). Thepump (17) is charged with the transport of the ATP solution, its outputrange extends from 0 to 16 ml/min. After that, the suspension is pumpedat an adjustable output of 0 to 32 ml/min in steps of 1 ml/min, andheated to 37° C. by passage through a heat exchanger. This isaccomplished by the pump (21) situated after the exchanger, so as towork on a portion of small tube which is not rigidified by a too lowtemperature. Then proportionally to the blood output and in a ratio of1/5th to 1/20th, the resealing product is mixed in the pouch (26) withthe blood heated to 37 degrees after passage through the heating pouch(22). A peristaltic pump (21) is used for this purpose, its output rangeextending from 0 to 16 ml/min.

The final step is a waiting phase for the product obtained in order toobtain the complete resealing of the red blood corpuscles. Thesuspension of red blood corpuscles is maintained for 15 to 30 min at thetemperature of 37° C. in the pouch of about 600 ml (26). After that, thewash II can then be performed.

EXAMPLE III Washing of the Red Blood Corpuscles After Resealing

The object of the second wash is to wash and resuspend, in plasma, thecellular suspension obtained after the lysis and resealing phase. Itcomprises the following steps:

filling the bowl and removing the solution, resuspending the red bloodcorpuscles, this solution containing essentially IHP, ATP and freehemoglobin.

removal of the stromata of cells.

suspension of the treated red blood corpuscles in a preserving solution,plasma or a solution of synthetic macromolecules.

Sequence of Operations

The suspension of cells treated by dialysis is introduced into the bowlalready used for the wash I at a rate of 100 cc/min, diluted withphysiological saline in a ratio of 1:2 and a centrifuge rotation speedof 5000 rpm. This phase is stopped after a volume of about 1 l has beenintroduced, or a detection of air in the small tube for supplying thebowl has occurred, an indication of the vacuity of the pouch. In thecase of a detection of air, the question arises as to whether to startthe washing or to wait for the availability of other blood to be washed.

The wash may then begin with the introduction, into the bowl, of aprogrammed volume of 9% NaCl solution, at a variable rate and for acentrifugation speed of 6000 rpm, under the control of the optic of thesmall tube. The output of the pump is controlled such that the liquidleaving the bowl is at a maximum hematocrit of the order of 1%,permitting the removal of stromata of cells while minimizing the loss ofred blood cells. This phase removes a portion of the free hemoglobinwithout, however, that which is present in the red blood corpusclesbeing actually affected.

The centrifuge is then stopped, then immediately restarted, so as tohomogenize the solution of suspension of red blood cells., beforerestarting, for a determined volume, the introduction of physiologicalsaline. Three steps of washing with 500 ml of physiological saline arethus performed.

This centrifuge stop and restart phase is repeated a second time, butonly for two steps of about 100 ml, the physiological saline beingreplaced with plasma.

The centrifuge is then stopped and the bowl is emptied into a transferpouch at a rate of 150 ml/min.

The washes I and II may correspond to a sequence of cycles (one ormore), a cycle corresponding to a set of phases and steps.

We claim:
 1. An apparatus for introducing at least one biologicallyactive substance into erythrocytes, comprising:a settling bowl forremoving plasma, leucocytes, platelets of blood, and adjustinghematocrit and controlling osmolarity of blood, and for obtainingerythrocytes suspended in a solution of a product to be internalized; alysis unit for cooling the erythrocyte suspension obtained from thesettling bowl and introducing the active substance into the coolederythrocyte suspension, the lysis unit having cooling means formaintaining the erythrocyte suspension at a temperature less than 10°C., a dialysis cartridge for receiving the cooled erythrocyte suspensionand a first collection pouch for receiving the erythrocyte suspensionprocessed by the dialysis cartridge, and means for introducing theactive substance into the first pouch along with the processederythrocyte suspension; and a resealing unit for heating the erythrocytesuspension treated with the active substance from the first pouch andintroducing a resealing product into the heated and treated erythrocytesuspension, the resealing unit having heating means for maintaining thetemperature of the treated erythrocyte suspension greater than 20° C., asecond collection pouch for receiving and holding the heated and treatederythrocyte suspension, and means for introducing the resealing productinto the second pouch along with the heated and treated erythrocytesuspension.
 2. An apparatus according to claim 1, wherein the lysis unitfurther comprises means for introducing and discharging a lysis bufferto and from the dialysis cartridge, where the cooling means in the lysisunit maintains the temperature of the erythrocyte suspension at 4° C. 3.An apparatus according to claim 2, wherein the dialysis cartridge hastwo compartments separated by at least one semipermeable membrane, thelysis buffer being introduced into one of the compartments and theerythrocyte suspension being introduced into the other compartment. 4.An apparatus according to claim 2, wherein the lysis unit furtherincludes means for cooling the lysis buffer before the lysis buffer isintroduced into the dialysis cartridge.
 5. An apparatus according toclaim 1, wherein the heating means in the resealing unit maintains thetemperature of the treated erythrocyte suspension at 37° C.
 6. Anapparatus according to claim 1, further comprising a peristaltic pumpfor transporting the treated erythrocytes from the first pouch to theresealing unit.
 7. An apparatus according to claim 1, further comprisinga first peristaltic pump for introducing the erythrocyte suspension intothe lysis unit and a second peristaltic pump for withdrawing the treatederythrocyte suspension from the resealing unit.
 8. An apparatusaccording to claim 1, wherein the first and second pouches are made fromplastic material.
 9. An apparatus according to claim 1, wherein thelysis unit further includes a baffled cooling pouch for receiving theerythrocyte suspension from the settling bowl, wherein the cooling pouchfeeds the cooled erythrocyte suspension to the dialysis cartridge andwherein the resealing unit further includes a baffled heating pouch forreceiving the treated erythrocyte suspension from the first pouch,wherein the heating pouch feeds the heated and treated erythrocytesuspension to the second pouch.
 10. An apparatus according to claim 1,wherein the first and second pouches are vertically situated and theerythrocyte suspension is introduced from the top and discharged fromthe bottom.
 11. An apparatus according to claim 1, wherein the dialysiscartridge is vertically situated and the cooled erythrocyte suspensionis introduced from the bottom and the processed erythrocyte isdischarged from the top.
 12. An apparatus according to claim 1, furthercomprising means for receiving and washing the heated and treatederythrocyte suspension from the second pouch.
 13. An apparatus accordingto claim 12, wherein the washing means is the settling bowl.
 14. Anapparatus according to claim 1, wherein the lysis unit further includesmeans for introducing another product into the dialysis cartridge. 15.An apparatus according to claim 1, further including means for purgingthe lysis unit or the resealing unit or both.
 16. An apparatus accordingto claim 1, wherein the lysis unit and the resealing unit are thermallyinsulated.
 17. An apparatus according to claim 1, wherein the heatingand cooling means each comprise a thermally conductive plate having arespective heating or cooling circuit on one side in contact therewith,wherein at least the dialysis cartridge and the first and secondcollection pouches of the lysis unit and the resealing unit areremovably attached to the opposite sides of the plates.
 18. An apparatusfor introducing at least one biologically active substances intoerythrocytes suspended in a solution of a product to be internalized,comprising:a) a lysis unit comprising:a cooling heat exchanger having aninlet for receiving the erythrocyte suspension and an outlet foroutputting the erythrocyte suspension at a temperature of about 4° C.; adialysis cartridge having an inlet for receiving the cooled erythrocytesuspension from the cooling heat exchanger and an outlet for outputtingthe erythrocyte suspension processed by the dialysis cartridge; meansfor introducing and discharging a lysis buffer solution into and out ofthe dialysis cartridge; a retardation pouch having an inlet and anoutlet, wherein the processed erythrocyte suspension from the dialysiscartridge is collected in the retardation pouch; means for introducingthe active substance into the retardation pouch along with the processederythrocyte suspension; and means for maintaining the temperature of theerythrocytes in the dialysis cartridge and the retardation pouch atabout 4° C.; and b) a resealing unit comprising:a heating heat exchangerhaving an inlet for receiving the treated erythrocyte solution from theretardation pouch and an outlet for outputting the treated erythrocytesuspension at a temperature of about 37° C.; a collection pouch havingan inlet and an outlet, wherein the heated and treated erythrocytesuspension from the heating heat exchanger is collected in thecollection pouch; means for introducing a resealing product into thecollection pouch along with the heated and treated erythrocytesuspension; and means for maintaining the temperature of the erythrocytesuspension in the collection pouch at about 37° C.
 19. An apparatusaccording to claim 18, wherein the cooling heat exchanger comprises abaffled cooling pouch for receiving the erythrocyte suspension, whereinthe cooling pouch feeds the cooled erythrocyte suspension to thedialysis cartridge and wherein the heating heat exchanger comprises abaffled heating pouch for receiving the treated erythrocyte suspensionfrom the retardation pouch, wherein the heating pouch feeds the heatedand treated erythrocyte suspension to the collection pouch.
 20. Anapparatus according to claim 19, wherein the lysis unit further includesmeans for cooling the lysis buffer before the lysis buffer is introducedinto the dialysis cartridge.